Targeting Enterococcus faecalis HMG-CoA reductase with a non-statin inhibitor

HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development of novel antibiotics. In this study, we report the crystal structures of HMGR from Enterococcus faecalis (efHMGR) in the apo and liganded forms, highlighting several unique features of this enzyme. Statins, which inhibit the human enzyme with nanomolar affinity, perform poorly against the bacterial HMGR homologs. We also report a potent competitive inhibitor (Chembridge2 ID 7828315 or compound 315) of the efHMGR enzyme identified by a high-throughput, in-vitro screening. The X-ray crystal structure of efHMGR in complex with 315 was determined to 1.27 Å resolution revealing that the inhibitor occupies the mevalonate-binding site and interacts with several key active site residues conserved among bacterial homologs. Importantly, 315 does not inhibit the human HMGR. Our identification of a selective, non-statin inhibitor of bacterial HMG-CoA reductases will be instrumental in lead optimization and development of novel antibacterial drug candidates.


Supplementary Figure 2 Domain swapping in efHMGR, pmHMGR and human HMGR
In the Class I human HMGR (green) and Class II efHMGR (magenta), the N-terminal domain of one monomer makes a cross over and interact with the second monomer. However, in Class II pmHMGR (blue), the N terminal domain double backs on itself and interact with the same monomer. In all cases, the second monomer is colored gray. and HMG-CoA. The nitrogen N4P, of the 1 st isopeptide bond is at a hydrogen bond distance to hydroxyl group of Ser88 (2.8Å). The CoA thioester sulphur atom lies close to the catalytic His-376 from the first helix of the flap domain. A hydrogen bond interaction is seen between Asn-95 side chain amide group, and the oxygen atom (O9P) of the pantothenate group (3.1Å).
A water mediated hydrogen bond interaction is also observed between the side chain amide group of Gln-359 and the oxygen (O2) of pantothenic group. The HMG/mevalonate binding pocket lies at the dimer interface in a relatively protected cleft and is conserved across all HMGRs. Two hydrogen bond interactions (2.5 Å and 2.9Å) are observed between the guanidino group of Arg-257 and the carboxylate group of HMG. The thioester bond is intact and the carbonyl oxygen of the thioester is rotated 180° as compared to the analogous pmHMGR structure (PDB: 1qax) in the presence of HMG-CoA and NAD + . In this configuration, the thioester oxygen lies is ~5Å away from the catalytic glutamate residue b. NADP + : The adenine ring of NADP + lies near the surface of the enzyme, stabilized by stacking interaction with Arg152 (3.5Å) and a hydrophobic interaction with Val323 (3.5Å).
The N1A atom and the 6-amino moeity of adenine interact with the main chain amide (3.3Å) and the side chain carboxyl group of Asp-179 (3.2 Å) respectively via hydrogen bonds. The pyrophosphate moiety hydrogen bonds with the main chain amide of Met-181(3.2Å) and Gly-182 (3.1Å) residues located in the small domain. The 2'-OH of the nicotinamide ribose group is stabilized by hydrogen bond interactions with side chain carboxyl oxygen of Asp-279 (2.8 Å). The nicotinamide-ribose-oxygen hydrogen bonds with the side chain amide group of Asn-184 (3.1Å). The carboxamide group of the nicotinamide ring is stabilized by hydrogen bond interactions with the side chain carboxamide moeity of Asn-213 (3.1Å and 2.8Å respectively). The interactions that stabilize the adenine-ribose-phosphate group of NADP + are discussed in the main text. In all the figures, cyan sphere represents water molecules, green dashes represent hydrogen bond interactions (between 2.3 to 3.3Å distance) and brick red spoked arcs represent hydrophobic interactions. Ligand bond is shown in purple, and atoms are shown as black for carbon, red for oxygen, yellow for sulpher and blue for nitrogen. Burkholderia cenocepacia, which is a gram-negative pathogenic bacterium is also included.
This alignment suggests that 315 binding residues (marked with blue asterix) are conserved across the pathogenic HMGRs. The semiconserved residue are marked with green asterix.

Interactions at the dimer-dimer interface of the apoenzyme
The tetramer in the asymmetric unit of the apoenzyme is comprised of two dimers (AB and CD). The dimer-dimer interface is observed primarily between the monomer A and C with a buried surface area of 390 Å 2 and is mediated by a calcium ion from the crystallization buffer (Supplementary Figure 1a). However, additional interactions are observed between monomers B and C, B and D, and A and D as well. The calcium ion present at the AC interface coordinates with the side-chain carboxyl oxygen of Glu-347 and side-chain hydroxyl group of Thr-343 from monomer A and the same set of residues from monomer C as well. Additional hydrogen bond interactions between residues Thr-343 (A) and Ala-341 In other prokaryotic HMGR enzymes including pmHMGR (PDB: 1qax) 32 , Streptococcus pneumoniae HMGR ( PDB: 5wpj) 18 , Delftia acidovorans HMGR (PDB: 6eeu) 26 and Burkholderia cenocepacia HMGR ( PDB: 6p7k) 19 , the ENQIS sequence is substituted by the ENVXG sequence which makes no such interactions. In pmHMGR, DaHMGR and BcHMGR, which swap only 16 residues , a proline residue (Pro-43/Pro-43/Pro-64) is present at the hinge region that precedes the ENVXG sequence. This proline residue introduces conformational constraints which force the rest of the N-terminal domain to fold back on itself and thereby prevents an entire domain swap. Hydrophobic interactions between Met-44/Leu-44/Leu-65 , Ala-47/Ala-47/Ala-68 and Val-54/Val-54/Val-65 further stabilize the partially swapped conformation in the other bacterial homologs (Figure 1d).